During FY07, we focussed primarily on four subprojects.[unreadable] [unreadable] (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (capsids) and e-antigen (unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into capsids of two different sizes, we obtained, in 1997, a cryo-EM density map in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. Our subsequent research helped delineate the path of the polypeptide chain. We went on to investigate the antigenic diversity of HBV by studying the modes of binding of six different monoclonal antibodies raised against capsids. In FY07, we completed a cryo-EM analysis of the binding to capsids of a receptor immunoglobulin from naive B-cells. Its binding affinity, measured by surface plasmon resonance, was found to be markedly lower than is typical of antibody-antigen interactions. Nevertheless, this Fab decorates capsids sufficiently to allow structural analysis. Its mode of binding is unlike that of any other Fab previously characterized in that a single molecule binds to two sites, 3 nm apart, on different spikes. One of these interactions is canonical, i.e. mediated by CDR loops and the other is non-canonical, involving a constant domain of the Fab.[unreadable] [unreadable] (2) Structure, Assembly, and Maturation of Herpesviruses. Herpesviruses are large complex DNA viruses, eight members of which cause diseases in humans, including skin diseases such as Kaposi's sarcoma which is prevalent in immunosuppressed AIDS patients. In this long-term project, we first defined the molecular anatomy of the herpesvirus capsid and then characterized the roles of its six major proteins in assembly. In these properties, herpesviruses differ from other animal viruses but closely resemble tailed bacteriophages, suggesting a distant evolutionary relationship. One major finding was our discovery of the HSV procapsid, which differs radically from the mature capsid in structure and stability. Maturation of the procapsid, controlled by the viral protease, is a target for antiviral drugs. New Results. UL25 and UL17 are two essential minor capsid proteins, implicated in DNA packaging and capsid maturation. We used cryo-EM to examine their binding to capsids. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique vertex-adjacent site that is not seen on unfilled capsids. Using also SDS-PAGE and mass spectrometry, we concluded that (i) the C-capsid-specific component is a heterodimer of UL25 and UL17; and (ii) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. Binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.[unreadable] [unreadable] (3) Assembly and Maturation of Bacteriophage Capsids. Our primary interest in capsid assembly lies in the massive changes that accompany their maturation. These transitions afford unique insights into regulatory conformational changes in protein complexes. We study maturation of several phages to exploit expedient aspects of each system. New Results: We investigated the thermodynamic basis of HK97 assembly by combining scanning calorimetry and cryo-electron microscopy. The procapsid, Prohead I, is a 45nm-diameter shell composed of 60 hexamers and 12 pentamers of gp5. Gp5 consists of an N-terminal delta-domain and gp5*: gp5* forms the contiguous shell from which clusters of delta-domains extend inwards. Prohead I exhibits a reversible endothermic transition at 53C. To capture its structural expression, we incubated Prohead I at 60C followed by rapid freezing and observed a capsid species 10% larger than Prohead I. Visible changes are confined to the gp5 hexamers. Their delta-domain clusters have disappeared, presumably through disordering. The gp5* hexamer rings assume the conformation observed in Expansion Intermediate I, a transition state of the normal proteolysis-induced maturation pathway. We infer that, at ambient temperatures, the hexamer delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53, this constraint is overcome. Pentamers, on the other hand, are stabler and resist this thermal perturbation. We also initiated a project to investigate capsid maturation of bacteriophage phi6, which has a tripartite genome of double-stranded RNA. It has been hypothesized that , in this system, capsid maturation proceeds in a stepwise fashion that is coupled to sequential packaging of the three segments, S, M and L. As a first step, we have calculated three-dimensional reconstructions of the prohead, i.e. the initial state.[unreadable] [unreadable] (4) Maturation and Pleiomorphy of Retroviruses. We participate in the intramural IATAP program to investigate HIV and AIDS, focussing on structural aspects of retroviruses. In FY07, we used our recently developed capability in cryo-electron tomography to investigate Rous Sarcoma Virus (RSV), the prototypic avian oncoretrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix (MA), capsid (CA) and nucleocapsid (NC) moieties. The CA subunits reassemble into cores housing the viral RNA and replication enzymes. The virions in our data set range from 105 to 170 nm in diameter. Their cores vary widely in shape: we observe angular cores, including some that are distinctively "coffin-shaped"; cores with continuous curvature including, rarely, fullerene cones; and tubular cores. The internal volume of a core and the density of encapsidated material vary with morphology. Angular cores are the most voluminous and densely packed; tubes and some curved cores contain relatively little material, raising the possibility that virions with tubular cores and some with curved cores may be non-infectious.